Development of a CAPS Marker and a LAMP Assay for Rapid Detection of Xylella fastidiosa Subsp. multiplex and Differentiation from X. fastidiosa Subsp. fastidiosa on Blueberry

Sumyya Waliullah; Dario Di Genova; Jonathan E Oliver; Md Emran Ali

doi:10.3390/ijms23041937

Development of a CAPS Marker and a LAMP Assay for Rapid Detection of Xylella fastidiosa Subsp. multiplex and Differentiation from X. fastidiosa Subsp. fastidiosa on Blueberry

Int J Mol Sci. 2022 Feb 9;23(4):1937. doi: 10.3390/ijms23041937.

Authors

Sumyya Waliullah¹, Dario Di Genova^{2

3}, Jonathan E Oliver¹, Md Emran Ali¹

Affiliations

¹ Department of Plant Pathology, University of Georgia, Tifton, GA 31793, USA.
² Department of Crop and Soil Sciences, University of Georgia, Tifton, GA 31793, USA.
³ Department of Agronomy, Food, Natural Resources, Animals and Environment, University of Padova, 35020 Legnaro, Italy.

Abstract

Bacterial leaf scorch (BLS), caused by Xylella fastidiosa (Xf), is a prevalent disease of blueberries in the southeastern United States. Initially, this disease was reported to be caused by X. fastidiosa subsp. multiplex (Xfm). However, a recent survey revealed the presence of another subspecies, X. fastidiosa subsp. fastidiosa (Xff), within naturally infected blueberry plantings in Georgia. Since knowledge regarding the origins of isolates causing Xf outbreaks can impact management recommendations, a routine method for identifying the pathogen at the subspecies level can be beneficial. Several detection strategies are available to identify Xf infection at the subspecies level. However, none of these have been developed for the routine and rapid differentiation of the blueberry-infecting Xf subspecies. Here, we developed two separate straightforward and rapid detection techniques, a cleaved amplified polymorphic sequence (CAPS) marker, and a loop-mediated isothermal amplification (LAMP) assay, targeting the RNA polymerase sigma-70 factor (rpoD) gene sequence of Xfm to discriminate between the two Xf subspecies infecting blueberry. With the CAPS marker, specific detection of Xfm isolates was possible from pure cultures, inoculated greenhouse-grown plant samples, and field infected blueberry samples by restriction digestion of the rpoD gene PCR product (amplified with primers RST31 and RST33) using the BtsI enzyme. The LAMP assay allowed for specific real-time amplification of a 204-bp portion of the XfmrpoD gene from both pure bacterial cultures and infected plant material using the Genie^® III system, a result further affirmed by gel electrophoresis and SYBR™ Green I DNA staining for visual observation. These detection strategies have the potential to greatly aid existing diagnostic methods for determining the distribution and prevalence of these Xf subspecies causing bacterial leaf scorch (BLS) in blueberries in the southeastern United States.

Keywords: CAPS marker; LAMP; bacterial leaf scorch (BLS); blueberry; subspecies detection.

MeSH terms

Blueberry Plants / microbiology*
DNA Primers / genetics
Genetic Markers / genetics*
Molecular Diagnostic Techniques / methods*
Nucleic Acid Amplification Techniques / methods*
Plant Diseases / microbiology*
Polymerase Chain Reaction / methods
Xylella / genetics*

Substances

DNA Primers
Genetic Markers

Supplementary concepts

LAMP assay
Xylella fastidiosa subsp. multiplex